Bottom Line:
We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain.Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process.

Background: Wnt proteins play roles in many biological processes, including axon guidance and cell migration. In the mammalian hindbrain, facial branchiomotor (FBM) neurons undergo a striking rostral to caudal migration, yet little is known of the underlying molecular mechanisms. In this study, we investigated a possible role of Wnts and the planar cell polarity (PCP) pathway in this process.

Results: Here we demonstrate a novel role for Wnt proteins in guiding FBM neurons during their rostral to caudal migration in the hindbrain. We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain. Wnt-coated beads chemoattracted FBM neurons to ectopic positions in an explant migration assay. The rostrocaudal FBM migration was moderately perturbed in Wnt5a mutant embryos and severely disrupted in Frizzled3 mutant mouse embryos, and was aberrant following inhibition of Wnt function by secreted Frizzled-related proteins. We also show the involvement of the Wnt/PCP pathway in mammalian FBM neuron migration. Thus, mutations in two PCP genes, Vangl2 and Scribble, caused severe defects in FBM migration. Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.

Conclusion: These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process. Molecules of the PCP pathway and the JNK and ROCK kinases also play a role in the FBM migration and are likely mediators of Wnt signalling.

Mentions:
In order to determine whether Wnt5a and/or Wnt7a might function as chemoattractants in vivo, we analysed their expression profiles in the mouse hindbrain. Whole-mount in situ hybridisation at E11.5 showed that in isolated neuroepithelial preparations, Wnt7a is expressed throughout the hindbrain, including a dorsal stripe and the floor plate (Figure 3A). Cryosections in situ-hybridised for Wnt7a mRNA showed that this expression was in the ventricular zone and generally in the neuroepithelium (Figure 3B–D). Intriguingly, whole-mount in situ hybridisation for Wnt5a showed a different pattern, with a caudalhigh to rostrallow gradient. This was a step gradient rather than a smooth gradient, with changes in expression level coinciding with rhombomere boundaries. There was little or no expression in r2/r3 and r4, apart from the floor plate, higher levels in r5/6 and then increased expression caudally, including a dorsal stripe (Figure 3E). Analysis of in situ-hybridised cryosections confirmed that Wnt5a forms a gradient in the hindbrain with higher levels caudally (Figure 3F–H). Taken together with the chemoattractant effect of Wnt protein in vitro, these expression patterns indicate that Wnt5a in particular could chemoattract FBM neurons along a rostrocaudal migration path in vivo, although Wnt7a could play an adjunct role. The rostrocaudal gradient of Wnt5a expression demonstrated here is consistent with previous data showing an attractant effect of r5/6 on FBM neurons in heterospecific mouse/chick grafting experiments [32].

Mentions:
In order to determine whether Wnt5a and/or Wnt7a might function as chemoattractants in vivo, we analysed their expression profiles in the mouse hindbrain. Whole-mount in situ hybridisation at E11.5 showed that in isolated neuroepithelial preparations, Wnt7a is expressed throughout the hindbrain, including a dorsal stripe and the floor plate (Figure 3A). Cryosections in situ-hybridised for Wnt7a mRNA showed that this expression was in the ventricular zone and generally in the neuroepithelium (Figure 3B–D). Intriguingly, whole-mount in situ hybridisation for Wnt5a showed a different pattern, with a caudalhigh to rostrallow gradient. This was a step gradient rather than a smooth gradient, with changes in expression level coinciding with rhombomere boundaries. There was little or no expression in r2/r3 and r4, apart from the floor plate, higher levels in r5/6 and then increased expression caudally, including a dorsal stripe (Figure 3E). Analysis of in situ-hybridised cryosections confirmed that Wnt5a forms a gradient in the hindbrain with higher levels caudally (Figure 3F–H). Taken together with the chemoattractant effect of Wnt protein in vitro, these expression patterns indicate that Wnt5a in particular could chemoattract FBM neurons along a rostrocaudal migration path in vivo, although Wnt7a could play an adjunct role. The rostrocaudal gradient of Wnt5a expression demonstrated here is consistent with previous data showing an attractant effect of r5/6 on FBM neurons in heterospecific mouse/chick grafting experiments [32].

Bottom Line:
We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain.Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process.

Background: Wnt proteins play roles in many biological processes, including axon guidance and cell migration. In the mammalian hindbrain, facial branchiomotor (FBM) neurons undergo a striking rostral to caudal migration, yet little is known of the underlying molecular mechanisms. In this study, we investigated a possible role of Wnts and the planar cell polarity (PCP) pathway in this process.

Results: Here we demonstrate a novel role for Wnt proteins in guiding FBM neurons during their rostral to caudal migration in the hindbrain. We found that Wnt5a is expressed in a caudal high to rostral low gradient in the hindbrain. Wnt-coated beads chemoattracted FBM neurons to ectopic positions in an explant migration assay. The rostrocaudal FBM migration was moderately perturbed in Wnt5a mutant embryos and severely disrupted in Frizzled3 mutant mouse embryos, and was aberrant following inhibition of Wnt function by secreted Frizzled-related proteins. We also show the involvement of the Wnt/PCP pathway in mammalian FBM neuron migration. Thus, mutations in two PCP genes, Vangl2 and Scribble, caused severe defects in FBM migration. Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins.

Conclusion: These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process. Molecules of the PCP pathway and the JNK and ROCK kinases also play a role in the FBM migration and are likely mediators of Wnt signalling.